The majority of HIV-1 replication and CD4+ T cell depletion occurs in lymphoid tissues. The reasons why endogenous HIV-1-specific CTLs are unable to fully suppress HIV-1 replication in lymphoid tissues are not understood. Interventions based on prevailing theories of CTL immune escape have unilaterally failed, and a new paradigm is urgently needed. We observed that virusspecific CTL fail to accumulate within lymphoid follicles of lymphoid tissues, where most HIV-1 replication is concentrated. Furthermore, we observed that follicular CD8+ cells have a noncytolytic effector memory phenotype, heightened PD-1 expression, and low density CD8 expression on tetramer-staining cells, suggesting that they may have impaired effector function. We hypothesize that lymphoid follicles are immune privileged sites due to 1) limited numbers of CXCR5+CD8+ cells that home to lymphoid follicles;and 2) impairments of effector function of the few CTL that enter lymphoid follicles relative to extrafollicular CTL. As a consequence, CTL exert antiretroviral activity primarily in extrafollicular regions of lymphoid tissues, but have little impact on virus replication within lymphoid follicles, resulting in persistent virus replication at those sites. The specific aims of this proposal are: 1) To determine the distribution of virus-specific CTL and virus-producing cells in lymphoid tissues of chronically SIVmac239-infected rhesus macaques and HIV-1-infected humans and investigate potential mechanisms underlying their distribution;2) To determine the functional properties of lentivirus-specific cells in follicular and extrafollicular compartments during chronic HIV-1 and SIV infection;and 3) To determine whether lentivirus-specific CTL activity is restricted to the extrafollicular compartment of lymphoid tissues in vivo. Virus-producing cells and virus-specific CTLs will be quantified in follicular and extrafollicular compartments of lymph nodes, spleen and GALT from SIV-infected macaques during acute and chronic stages of infection, and after CD8+ cell depletion using in situ hybridization, in situ tetramer staining, and immunostaining techniques. Functions of follicular CD8+ T cells will be determined by in situ staining of tissues with tetramers and antibodies to effector molecules, as well as by intracellular cytokine staining assays on cells from disaggregated lymphoid tissues stimulated with lentivirus peptides. Information on the frequency and function of follicular CD8 T cells within lymphoid tissues will yield critical insight into mechanisms underlying persistence of HIV-1 replication. If the proposed studies confirm our hypothesis, these data would support a therapeutic approach to enhance HIV-1 containment through augmentation of functional virus-specific CTL that home to lymphoid follicles.